Capillary malformation-arteriovenous malformation (CM-AVM) is an autosomal dominant blood vascular disorder in humans that is caused by inherited inactivating mutations of the RASA1 gene. Lymphatic vessel (LV) abnormalities such as chylothorax, lymphedema and impaired LV drainage have also been reported in CM- AVM. RASA1 acts as a negative regulator of the Ras small GTPase during the course of growth factor receptor intracellular signal transduction. However, how RASA1 mutations lead to the development of LV abnormalities in CM-AVM is not well understood. A long-term goal of the King laboratory is to understand the role of the Ras signaling pathway in LV development and function. The overall objective of this application, which is consistent with this long-term goal, s to determine the cellular, molecular, and genetic basis of LV abnormalities in CM- AVM. Our central hypothesis is that LV abnormalities in CM-AVM arise as a consequence of deficient expression of RASA1 in LV valve cells and that dysregulated activation of specific Ras effectors in this cell type results in impaired valve development and function. Furthermore, we hypothesize that somatic inactivation of the wild type RASA1 allele at an early point in embryogenesis is necessary for the development of LV abnormalities in CM-AVM. The rationale for these studies is that they will inform as to the most effective means of drug therapy of LV abnormalities in CM-AVM and other diseases that are caused by hyper-activation of Ras. We propose to test the central hypothesis through the pursuit of three independent specific aims. In the first aim, we will use an inducible RASA1-deficient mouse model of LV abnormalities in CM-AVM to examine the influence of Rasa1 gene disruption upon LV valve development and function and its relationship to LV hyperplasia. In the second aim, we will use the same mouse model to examine which intracellular signaling cascades downstream of Ras drive the development of LV abnormalities in this disease. In the third aim, we will perform genetic analyses upon biopsy samples obtained from CM-AVM patients with LV abnormalities to identify which type(s) of somatic genetic events act in concert with the germline RASA1 mutation to give rise to LV lesions. The proposed studies are innovative because of the use of novel methods to assess LV valve function and mechanisms of RASA1 gene inactivation. The proposed studies are significant because they are expected to lead to a means of therapy for LV disease in CM-AVM. They are also expected to advance the field of LV research in general.